WO2010083327A2 - Mesure de taux de frataxine - Google Patents

Mesure de taux de frataxine Download PDF

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Publication number
WO2010083327A2
WO2010083327A2 PCT/US2010/021067 US2010021067W WO2010083327A2 WO 2010083327 A2 WO2010083327 A2 WO 2010083327A2 US 2010021067 W US2010021067 W US 2010021067W WO 2010083327 A2 WO2010083327 A2 WO 2010083327A2
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WIPO (PCT)
Prior art keywords
frataxin
microsphere
antibody
human
polypeptide
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PCT/US2010/021067
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English (en)
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WO2010083327A3 (fr
Inventor
Devin Oglesbee
Dietrich Matern
Grazia Isaya
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Mayo Foundation For Medical Education And Research
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Priority to US13/143,304 priority Critical patent/US20110269246A1/en
Publication of WO2010083327A2 publication Critical patent/WO2010083327A2/fr
Publication of WO2010083327A3 publication Critical patent/WO2010083327A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics
    • G01N2800/385—Congenital anomalies

Definitions

  • This document relates to methods and materials involved in measuring levels of a frataxin polypeptide present in a biological sample.
  • this document provides methods and materials related to the use of anti-frataxin antibody-bound microspheres and biotinylated anti-frataxin antibodies to measure the levels of a frataxin polypeptide in a biological sample from a mammal ⁇ e.g., a newborn human).
  • FRDA Friedreich ataxia
  • This document relates to methods and materials involved in measuring levels of a frataxin polypeptide present in a biological sample.
  • methods and materials related to the use of anti-frataxin antibody-bound microspheres and biotinylated anti- frataxin antibodies to measure the levels of a frataxin polypeptide in a biological sample from a mammal e.g., a newborn human
  • the methods and materials provided herein can be used for diagnosis, identification of carrier status, and treatment monitoring of Friedreich ataxia patients and their families.
  • the methods and materials provided herein can be used to determine levels of a frataxin polypeptide at the same time as levels of other disease-associated polypeptides, for universal newborn screening for FRDA.
  • this document features a method for assessing levels of a frataxin polypeptide in a mammal.
  • the method comprises contacting an anti-frataxin antibody conjugated microsphere with a biological sample from a mammal, under conditions wherein a frataxin polypeptide present in the sample binds the microsphere, thereby forming a frataxin-microsphere complex, contacting the frataxin-microsphere complex with a detector-conjugated anti-frataxin antibody under conditions wherein the detector- conjugated anti-frataxin antibody binds the frataxin-microsphere complex, and quantifying the detector bound to the complex, thereby measuring levels of the frataxin polypeptide present in the sample.
  • the mammal can be a human.
  • the human can be a newborn.
  • the biological sample can be a biological fluid.
  • the biological fluid can be eluted from a dried blood sample.
  • the dried blood sample can be on filter paper.
  • the microsphere can comprise a fluorochrome.
  • the microsphere can be carboxylated.
  • the anti-frataxin antibody-conjugated microsphere can comprise a monoclonal antibody.
  • the detector can be biotin.
  • the quantification step can comprise contacting the complex with a composition comprising a streptavidin conjugate.
  • the streptavidin conjugate can be streptavidin-R-phycoerythrin.
  • the quantification step can comprise analysis on a flow cytometer.
  • this document features a method of assessing a human for
  • the method comprises contacting an anti-frataxin antibody conjugated microsphere with a biological sample from a human, under conditions wherein a frataxin polypeptide present in the sample binds the microsphere, thereby forming a frataxin- microsphere complex, contacting the frataxin-microsphere complex with a detector- conjugated anti-frataxin antibody under conditions wherein the detector-conjugated anti- frataxin antibody binds the frataxin-microsphere complex, determining whether or not a biological fluid from the human contains a decreased level of a frataxin polypeptide, and communicating a diagnosis of Friedreich ataxia if a decreased level of a frataxin polypeptide is determined, thereby diagnosing the human with Friedreich ataxia.
  • the human can be a newborn.
  • the sample can be a dried blood sample.
  • the microsphere can comprise a fluorochrome.
  • the microsphere can be carboxylated.
  • the anti-frataxin antibody-coupled microsphere can comprise a monoclonal antibody.
  • the detector can be biotin.
  • the quantification step can comprise contacting the complex with a composition comprising a streptavidin conjugate.
  • the streptavidin conjugate can be streptavidin-R- phycoerythrin.
  • the quantification step can comprise analysis on a flow cytometer. In another aspect, this document features an article of manufacture.
  • the article of manufacture comprises a vial containing anti-frataxin antibody-coupled microspheres, and a vial containing biotinylated anti-frataxin antibodies.
  • the article of manufacture can comprise a vial containing purified human frataxin.
  • Figure 1 is a schematic representation of an exemplar microsphere-conjugated sandwich immunoassay for measurement of the level of a frataxin polypeptide present in a biological sample.
  • Figure 2 is a graph plotting the amount of frataxin polypeptide (ng/punch) for an infant with FRDA and normal infants.
  • Figure 3 is a graph plotting the amount of frataxin polypeptide (ng/punch) present in samples from 23 unaffected adults and 51 adults with FRDA.
  • Figure 4 is a graph plotting the percent of ceruloplasmin polypeptide eluted from four newborn dried blood spots having less than one month of storage and five newborn dried-blood spots having greater than one year of storage.
  • This document relates to methods and materials involved in measuring levels of a human frataxin polypeptide.
  • this document provides methods and materials related to the use of a capture sandwich immunoassay featuring anti-frataxin antibody- bound microspheres and anti-frataxin antibody-bound detection molecules, to measure the levels of a frataxin polypeptide present in a biological sample (e.g., a dried blood sample) from a mammal (e.g., a newborn human). Any mammal can be assessed for reduced frataxin polypeptide levels using the methods and materials provided herein.
  • a human, mouse, cat, dog, or horse can be evaluated by assessing the levels a frataxin polypeptide in a biological sample to determine whether or not the mammal has FRDA.
  • a human suspected to have FRDA can be assessed.
  • a human between the ages of about 3-14 days old can be assessed.
  • a human older than about 14 days old can be assessed.
  • a human less than about 3 days old, e.g., a newborn infant can be assessed.
  • a frataxin polypeptide from a biological sample, such as a dried blood sample, biological fluid, or tissue.
  • biological fluids include, without limitation, serum, plasma, and cerebrospinal fluid.
  • a biological fluid can be obtained from a mammal by any appropriate method. For example, blood can be collected from heel or finger punctures by using single-use lancing devices.
  • the level of frataxin polypeptides can be detected using the methods and materials provided herein.
  • a capture-sandwich immunoassay can include using an anti-frataxin polypeptide antibody.
  • An anti-frataxin polypeptide antibody can be labeled for detection.
  • an anti-frataxin polypeptide antibody can be labeled with a radioactive molecule, a fluorescent molecule, or a bioluminescent molecule.
  • Frataxin polypeptides can also be detected indirectly using a labeled antibody that binds to an anti-frataxin polypeptide antibody that binds to a frataxin polypeptide.
  • An anti- frataxin polypeptide antibody can bind to a frataxin polypeptide with an affinity of at least 10 4 mol 1 (e.g., at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 mol 1 ).
  • An anti- frataxin antibody can be a polyclonal or monoclonal antibody.
  • monoclonal anti-human frataxin polypeptide antibodies are commercially available, e.g., from MitoSciences Inc., clone # 17A I I AC7.
  • a frataxin polypeptide 14 can be sandwiched by a microsphere 10 conjugated to an antibody 12, and detector-conjugated antibody 16.
  • a detector 18 can bind a reporter compound 20 to produce a signal that can be detected and quantified.
  • a microsphere can be conjugated to a frataxin polypeptide- specific antibody. Any appropriate method can be used to conjugate a microsphere to an anti-frataxin antibody, e.g., incubation. Conditions for antibody conjugation can be varied depending on the affinity of the antibody for a frataxin polypeptide, the optimal antibody concentration on the microsphere, and the surface chemistry of the microsphere.
  • a microsphere can be coated to reduce non-specific binding of serum proteins, e.g., a car boxy I tiled microsphere.
  • a microsphere can have defined spectral properties. For example, a microsphere can emit fluorescence in response to laser line excitation.
  • Microsphere bound-frataxin polypeptide can be quantified indirectly using a defector-conjugated antibody.
  • an anti-frataxin antibody can be used as a detector antibody, such as polyclonal antibody HFxn Ab251S.
  • the detector antibody can be labeled wUh a radioactive molecule, a fluorescent molecule, a bioluminescent molecule, an enzyme, or a ligand.
  • a detector antibody can be biotinylated, and levels of frataxin polypeptides in a biological sample can be measured by detecting fluorescence from an avidin conjugate, e.g., streptavidin-R-phycoerythrin.
  • the concentration of frataxin polypeptides can be assessed by any instrument capable of analyzing a solid phase sandwich immunoassay.
  • captured frataxin polypeptides can be measured by instruments capable of quantifying a detector-conjugated antibody, e.g. , flow cytometer.
  • materials described herein can be analyzed by a flow cytometer such as a Luminex 100TM or 200TM instrument.
  • the level of a frataxin polypeptide in a biological fluid from a mammal is determined, then the level can be compared to a median level or a cutoff level and used to determine whether or not the mammal has FRDA. If it is determined that a biological fluid from a mammal contains a reduced or decreased level of a frataxin polypeptide, then the mammal can be classified as having FRDA. For example, if it is determined that a biological fluid from a human infant (e.g., dried-blood spot sample) contains less than 0.2 (e.g., less than 0.15, less than 0.1, or less than 0.05) ng of a frataxin polypeptide per punch, then the human infant can be classified as having FRDA.
  • a biological fluid from a human infant e.g., dried-blood spot sample
  • a biological fluid from a human adult e.g., dried- blood spot sample
  • a biological fluid from a human adult contains less than 0.06 (e.g., less than 0.05, less than 0.04, or less than 0.03) ng of a frataxin polypeptide per punch
  • the human adult can be classified as having FRDA. See, e.g., Figure 3.
  • the level of a frataxin polypeptide in a biological fluid can be used in combination with one or more other factors to determine whether or not a mammal has FRDA.
  • a frataxin polypeptide level in a biological fluid can be used in combination with a gait or reflex test.
  • assessing the level of a frataxin polypeptide in a biological fluid can include using the level of an internal reference polypeptide (e.g., ceruloplasmin polypeptides) to confirm the quality of the sample. See, e.g., Figure 4. For example, if the elution of an internal reference polypeptide is below a permissible threshold level, then the quality of the frataxin polypeptide measurement can be insufficient to report and an additional sample or specimen may be needed for analysis.
  • a level of a frataxin polypeptide that is greater than 0.08 ng/punch can be considered normal, a level of a frataxin polypeptide that is between 0.08 and 0.06 ng/punch can be considered intermediate, and a level of a frataxin polypeptide that is less than 0.06 ng/punch can be considered a decreased level.
  • a mammal e.g., a human infant or adult
  • This document also provides methods and materials to assist medical or research professionals in determining whether or not a mammal has FRDA.
  • Medical professionals can be, for example, doctors, nurses, medical laboratory technologists, and pharmacists. Research professionals can be, for example, principal investigators, research technicians, postdoctoral trainees, and graduate students.
  • a professional can be assisted by (1) determining the level of a frataxin polypeptide in a biological fluid, and (2) communicating information about the level to that professional.
  • a medical professional can take one or more actions that can affect patient care. For example, a medical professional can record the level of a frataxin polypeptide in a patient's medical record. In some cases, a medical professional can record a diagnosis of FRDA, or otherwise transform the patient's medical record, to reflect the patient's medical condition. In some cases, a medical professional can review and evaluate a patient's entire medical record, and assess multiple treatment strategies, for clinical intervention of a patient's condition.
  • a medical professional can initiate or modify treatment for FRDA symptoms after receiving information regarding a patient's frataxin polypeptide levels.
  • a medical professional can compare previous reports of frataxin polypeptide levels with the recently communicated frataxin polypeptide level, and recommend a change in therapy.
  • a medical professional can enroll a patient in a clinical trial for novel therapeutic intervention of FRDA symptoms.
  • a medical professional can elect waiting to begin therapy until the patient's symptoms require clinical intervention.
  • a medical professional can communicate the levels of a frataxin polypeptide to a patient or a patient's family.
  • a medical professional can provide a patient and/or a patient's family with information regarding FRDA, including treatment options, prognosis, and referrals to specialists, e.g., cardiologists and/or genetic counselors.
  • a medical professional can provide a copy of a patient's medical records to communicate the levels of a frataxin polypeptide, to a specialist.
  • a research professional can apply information regarding a subject's frataxin polypeptide levels to advance FRDA research. For example, a researcher can compile data on frataxin polypeptide levels with information regarding the efficacy of a drug for treatment of FRDA symptoms to identify an effective treatment.
  • a research professional can obtain a subject's frataxin polypeptide levels to evaluate a subject's enrollment, or continued participation in a research study or clinical trial.
  • a research professional can classify the severity of a subject's condition, based on the levels of a frataxin polypeptide.
  • a research professional can communicate a subject's frataxin polypeptide level to a medical professional.
  • a research professional can refer a subject to a medical professional for clinical assessment of FRDA, and treatment of FRDA symptoms.
  • Any appropriate method can be used to communicate information to another person (e.g., a professional).
  • information can be given directly or indirectly to a professional.
  • a laboratory technician can input frataxin polypeptide levels into a computer-based record.
  • information is communicated by making an physical alteration to medical or research records.
  • a medical professional can make a permanent notation or flag a medical record for communicating a diagnosis to other medical professionals reviewing the record.
  • any type of communication can be used to communicate the information. For example, mail, e-mail, telephone, and face-to-face interactions can be used.
  • the information also can be communicated to a professional by making that information electronically available to the professional.
  • the information can be communicated to a professional by placing the information on a computer database such that the professional can access the information.
  • the information can be communicated to a hospital, clinic, or research facility serving as an agent for the professional.
  • a calibration curve is generated with each analytical batch and is found in column 1 on the plate.
  • a 7-point calibration curve is generated and analyzed (Table 2). The calibration is acceptable if the standard curve of the dilutions has R 2 > 0.9900. Table 2
  • assay buffer filtered PBS, 1% BSA, pH 7.4, 0.02% Sodium Azide
  • Column 1 is reserved for the standards.
  • the first two wells and last two wells on the plate are reserved for the controls.
  • the plate is covered with an EZ Seal plate sealer and placed on a MaxQ ® orbital shaking incubator at 37° C for 3 hours.
  • Frataxin antibody-coupled microspheres (Carboxylated Microspheres Region # 8, Luminex, Inc; clone 17Al 1AC7, MitoSciences Inc.) are suspended by vortex and sonication for about 20 seconds, and diluted to a final concentration of 4000 microspheres / 50 ⁇ L in assay buffer.
  • a 1.2 ⁇ m Millipore filter plate is pre-wetted with 100 ⁇ L/well of assay buffer aspirated by vacuum manifold.
  • a 50 ⁇ L aliquot of the microsphere mixture is pipetted into the appropriate wells of the filter plate.
  • 50 ⁇ L aliquots of the standards and controls are pipetted into the appropriate wells.
  • 50 ⁇ L is transferred from all eluted patient wells to the appropriate wells on a 96-well Millipore filter plate.
  • the reactions are mixed gently by pipetting up and down several times with a multi-channel pipettor.
  • the filter plate is covered and incubated for 90 minutes at 37° C at 225 rpm on the Barnstead MaxQ ® orbital shaking incubator.
  • the supernatant is aspirated by vacuum manifold.
  • Each well is washed twice with 100 ⁇ L of assay buffer and aspirated by vacuum manifold.
  • the microspheres are resuspended in 50 ⁇ L of assay buffer by gently pipetting up and down five times with a multi-channel pipettor.
  • the HFxn Ab2518 antibodies are biotinylated for use as detection antibodies (EZ Link Micro Sulfo-NHS-LC-Biotinylation Kit, Pearce, Inc.). 50 ⁇ L of the diluted biotinylated detection antibody are added to each well, and mixed gently.
  • the filter plate is covered and incubated for 90 minutes at 37° C at 225 rpm on the Barnstead MaxQ ® orbital shaking incubator.
  • the supernatant is aspirated by vacuum manifold.
  • Each well is washed twice with 100 ⁇ L of assay buffer and aspirated by vacuum manifold.
  • the microspheres are resuspended in 50 ⁇ L of assay buffer, and 50 ⁇ L streptavidin-R-phycoerythrin (SAPE) (4 ⁇ g/mL) is added to each well.
  • SAPE streptavidin-R-phycoerythrin
  • the filter plate is covered and incubated for 30 minutes at room temperature on a plate shaker.
  • the supernatant is aspirated by vacuum manifold.
  • Each well is washed twice with 100 ⁇ L of assay buffer and aspirated by vacuum manifold.
  • microspheres After washing and resuspending in 100 ⁇ L of assay buffer, 50-75 ⁇ L of the microsphere solution is analyzed on the Luminex ® analyzer according to the system manual. Briefly, microspheres are excited by a 633 nm laser, and emit fluorescence that is detected by two avalanche photo diodes (APD). The analyte reporter (streptavidin-R- phycoerthrin) is excited by a 532 nm laser, and fluorescence is detected by a photomultiplier tube (PMT)) ( Figure 1).
  • PMT photomultiplier tube
  • SAPE fluorescence the concentration of frataxin polypeptides in each well is determined. These data are correlated to the concentration of the calibrator. The concentration of the calibrator is converted from ng/well to the final reporting units of ng/ ⁇ L blood. Results below the normal range are called back to the requesting physician by the Genetic Counselor or consultant on-call.
  • Filter paper card grade 903
  • the paper cards are dried at room temperature, in a horizontal position for three or more hours. Specimens are stored at ambient, refrigerated or frozen conditions, but not for more than 48 hours at temperatures exceeding 25°C.
  • Calibrators are prepared from purified human Ceruloplasmin (CPl 1,
  • assay buffer filtered PBS, 1% BSA, pH 7.4, 0.02% Sodium Azide
  • Column 1 is reserved for the standards.
  • the first two wells and last two wells on the plate are reserved for the controls.
  • the plate is covered and placed on an orbital shaker at ambient temperature for 3 hours.
  • Anti- frataxin antibody-coupled microspheres (Carboxylated Microspheres Region # 11, Luminex, Inc; antibody clone 17A11AC7, MitoSciences Inc.) and anti-Ceruloplasmin antibody-coupled microspheres (Carboxylated Microspheres Region # 8, Luminex, Inc; antibody clone CR6010RP, Cortex BioChem) are suspended by vortex and sonication for about 20 seconds, and diluted to a final concentration of 3000 microspheres / 50 ⁇ L in assay buffer.
  • a 1.2 ⁇ m Millipore filter plate is pre -wetted with 100 ⁇ L/well of assay buffer aspirated by vacuum manifold.
  • a 50 ⁇ L aliquot of the microsphere mixture is pipetted into the appropriate wells of the filter plate.
  • 50 ⁇ L aliquots of the standards and controls are pipetted into the appropriate wells.
  • 50 ⁇ L is transferred from all eluted patient wells to the appropriate wells on a 96-well Millipore filter plate.
  • the reactions are mixed gently by pipetting up and down several times with a multi-channel pipettor.
  • the filter plate is covered and incubated for 90 minutes at ambient temperature at >200 rpm on the orbital shaker. After incubation, the supernatant is aspirated by vacuum manifold.
  • Each well is washed three times with 100 ⁇ L of wash buffer (filtered PBS, 0.05& Tween 20, pH 7.4) and aspirated by vacuum manifold.
  • the microspheres are resuspended in 50 ⁇ L of assay buffer by gently pipetting up and down five times with a multi-channel pipettor.
  • Anti- Frataxin antibodies (Clone HFxn Ab2518, Mayo Clinic) and anti-ceruloplasmin antibodies (clone WD 1.1, Mayo Clinic) are biotinylated for use as detection antibodies (EZ Link Micro Sulfo-NHS-LC-Biotinylation Kit, Pearce, Inc.). 50 ⁇ L of the combined diluted biotinylated detection antibody are added to each well, and mixed gently.
  • the filter plate is covered and incubated for 90 minutes at ambient at >200 rpm on the orbital shaker.
  • the supernatant is aspirated by vacuum manifold.
  • Each well is washed twice with 100 ⁇ L of wash buffer and aspirated by vacuum manifold.
  • the microspheres are resuspended in 50 ⁇ L of assay buffer, and 50 ⁇ L streptavidin-R-phycoerythrin (SAPE) (1 ⁇ g/mL) is added to each well.
  • SAPE streptavidin-R-phycoerythrin
  • the filter plate is covered and incubated for 30 minutes at room temperature on a plate shaker.
  • the supernatant is aspirated by vacuum manifold.
  • Each well is washed twice with 100 ⁇ L of wash buffer and aspirated by vacuum manifold.
  • microspheres After washing and resuspending in 100 ⁇ L of assay buffer, 80 ⁇ L of the microsphere solution is analyzed on the Luminex analyzer according to the system manual. Briefly, microspheres are excited by a 633 nm laser, and emit fluorescence that is detected by two avalanche photo diodes (APD). The analyte reporter (streptavidin-R- phycoerthrin) is excited by a 532 nm laser, and fluorescence is detected by a photomultiplier tube (PMT)) ( Figure 1).
  • PMT photomultiplier tube
  • SAPE fluorescence the concentration of frataxin polypeptides in each well is determined. These data are correlated to the concentration of the calibrator. The concentration of the calibrator is converted from ng/well to the final reporting units of ng/ ⁇ L of blood or ng/punch. The co-analysis of the protein ceruloplasmin, provides an additional quality control parameter to evaluate the elution of the proteins from the blood spots. Low
  • Frataxin results combined with low Ceruloplasmin results would indicate poor elution or improper handling of the blood spot rather than a below normal Frataxin result. Frataxin results below the normal range are called back to the requesting physician by the Genetic Counselor or consultant on-call.
  • a single retrospective, newborn screening dried-blood spot was obtained with consent from an affected FRDA infant and was processed, along side three different retrospective newborn screening dried-blood spots obtained from unaffected infants (samples: Normal 1, 2, and 3), using a frataxin quantification, dried blood spot assay.
  • the quantity of frataxin polypeptide (ng) per 3 mm blood-spot-punch is shown in
  • Example 5 Additional measurements to confirm decreased frataxin polypeptide levels
  • the percent of ceruloplasmin elution can be determined in order to identify whether the low frataxin level is specific (i.e., as expected for a FRDA patient), or non-specific as observed in compromised dried-blood spot samples (e.g., samples stored for > 1 year).

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Abstract

L'invention concerne des méthodes et des matériaux permettant de mesurer le taux d'un polypeptide de frataxine présent dans un prélèvement biologique. L'invention concerne, par exemple, des méthodes et des matériaux permettant d'utiliser des microsphères liées à un anticorps anti-frataxine et des anticorps anti-frataxine biotinylés pour mesurer le taux d'un polypeptide de frataxine dans un prélèvement biologique provenant d'un mammifère (un nouveau-né humain, par exemple).
PCT/US2010/021067 2009-01-14 2010-01-14 Mesure de taux de frataxine WO2010083327A2 (fr)

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